Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aggregation of platelets by the antibiotic, ristocetin, requires a plasma cofactor (VIII:vWF) and one or more specific binding sites on the platelet membrane. The interaction between VII:vWF and the platelet was examined using VIII:vWF labelled with 125I. In the presence of ristocetin (1.5 mg/ml), from 70 to 90% of the 125I-VIII:vWF became platelet-bound. By contrast, only 21% was bound with thrombin (2.5 microgram/ml), and 2.2% with buffer alone. Fractionation of the platelets revealed that peak radioactivity was present in the membrane fraction. Treatment of ristocetin-reacted platelets with either chymotrypsin, 100 microgram/ml, or trypsin, 75 microgram/ml, resulted in the partial release of the membrane-bound radioactivity. It is concluded that VIII:vWF binds to the platelet membrane in the presence of ristocetin.
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PMID:Platelet-binding of the von Willebrand factor. 30 91

Galline, a protamine of domestic fowl, was obtained by two preparation procedures from the semen of a strain of White Plymouth Rock and submitted to fractionation by column chromatography on Bio-Gel CM-30. In the first procedure the specimen prepared from sperm heads was purified by the use of distilled water and dilute acetic acid and fractionated into almost eight fractions (G-I-G-VIII) in the same way as the specimen from a strain of New Hampshire (1,2). No difference could be found between galline specimens from the two different strains based on the amino acid and terminal analyses of each fraction. The specimen of galline from sperm heads purified with 1% citric acid (the second procedure) was composed of only one component, which was isolated as a single peak. The smaller fractions, G-I-G-VII, were found to be derived from G-VIII by the action of trypsin-like protease contained in the extract of sperm heads with 1% citric acid. This enzyme seems to originate in the acrosome of fowl spermatozoa. Consequently, it is concluded that intact galline is composed of only one molecular species and its total amino acid sequence is represented by the completed formula of G-VIII as shown in the preceding paper (4).
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PMID:Studies on a protamine (galline) from fowl sperm. 4. Degradation of galline by trypsin-like protease of fowl sperm heads. 99 42

A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.
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PMID:Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely. 153 49

The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70 degrees C for 60 min but was inactivated at 85 degrees C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacillus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50% of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
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PMID:Stability of the recombinant hepatitis B core antigen. 162 88

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.
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PMID:Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex. 170 66

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.
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PMID:Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13. 172 85

The effects of experimental cryptorchidism on seminiferous tubule secretions and interstitial cell testosterone production were studied in vitro. Spent media obtained from incubations of seminiferous tubules (SMST) from cryptorchid rats caused a significant increase in testosterone production when added to interstitial cells isolated from intact rats. The previously noticed inhibitory activity of the SMST from stages VIII-XI of the spermatogenic epithelial cycle gradually disappeared after the induction of experimental cryptorchidism. SMST obtained from both sham-operated or cryptorchid rats stimulated basal testosterone production when added to interstitial cells from cryptorchid rats. SMST from rats had been cryptorchid for 7, 14 and 28 days stimulated testosterone production when added to interstitial cells prepared from intact animals. Seminiferous tubules from cryptorchid rats therefore appear to be the source of a heat stable, trypsin-resistant factor with an apparent molecular weight of between 5000 and 10,000 daltons which stimulates testosterone production when added to interstitial cells in vitro. Its activity could not be blocked by an LRH antagonist. This factor enhances both basal and LH-stimulated secretion of testosterone in contrast to the inhibitory activity which involves only a partial blockade of LH-dependent steroidogenesis.
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PMID:Regulation of interstitial cell function by seminiferous tubules in intact and cryptorchid rats. 287

The factors responsible for the lability of factor VIII coagulant activity (VIII:C) and factor VIII coagulant antigen (VIII:CAg) are poorly understood. In this study the VIII:C and VIII:CAg are studied after incubation with plasmin, trypsin or alpha-chymotrypsin. Both isolated human VIII:CAg and VIII:CAg associated with factor VIII-related antigen (VIII R:Ag) are evaluated. The antigenic sites of the VIII:CAg are somewhat more stable to the action of these enzymes than the functional activity, although both follow a generally parallel degradation. A biphasic decay curve is seen in the initial time points. No stabilization of the functional or antigenic reactivity is observed in the presence of the VIII R:Ag. Lower concentrations of each enzyme cause an initial rise in the factor VIII:C in the presence of VIII R:Ag, but not in the isolated VIII:CAg. Higher concentrations of alpha-chymotrypsin cause activation of VIII:C and a slight decrease in the VIII:CAg values in both preparations. These enzymes may play a modulating role in the coagulation cascade through the activation and degradation of VIII:C and VIII:CAg.
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PMID:Degradation of factor VIII coagulant antigen by proteolytic enzymes. 293 56

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.
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PMID:The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis. 301 77

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.
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PMID:Identification of a factor IX/IXa binding protein on the endothelial cell surface. 303 54


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